Many human recombinant proteins form inclusion body or non-native form when expressed in E. coli and so denaturing conditions, such as the use of 8 M urea, have to be used to purify protein, which may cause trouble during protein purification and preparation of purified protein before antibody production than protein purified as soluble protein or under native conditions. For example, the procedures to purify proteins in inclusion body are more complicated than that for purifying soluble proteins. Moreover, if denaturing conditions are used to purify proteins, removal of urea in purified protein before antibody production is very troublesome, such as the use of lengthy buffer-exchange processes with concentrators, or even sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) purification. Furthermore, very often proteins purified under denaturing conditions may become aggregated or form precipitation once the denaturing agent is removed and the protein is concentrated, which cause problems by reducing amount of protein yield available for antibody production and subsequent affinity purification of antibody. Attempts to refold such purified protein back to native form often do not work well or are difficult. On the other hand, only soluble recombinant protein, but not aggregated protein, can be used for pharmaceutical purposes and to study the biochemical properties, biological functions, and crystal structure.
Although antibodies generated against non-native form of protein or synthetic peptide for specific epitopes can be used for the purpose of Western blot analysis, the use of native protein as the antigen might produce more useful polyclonal antibody against dominant epitopes presented by natively folded protein. Moreover, antibody that can recognize epitopes the bided structure of mature protein would be needed for immunoprecipitation assay. Multiple try-and-error may be needed when only aiming at specific epitopes by synthetic peptides. Moreover, denatured recombinant proteins are often in aggregated or precipitated form, which cause low yield and high cost if using soluble fraction to immunize rabbits and is not suitable for further purification of antibody by affinity column.
Although administration of exogenous coenzyme Q10 has been extensively used, the biosynthesis for endogenous coenzyme Q10 and mechanisms for coenzyme Q10 deficiency diseases in humans are poorly studied. The lack of validated commercial antibodies and soluble purified recombinant proteins for human PDSS and COQ proteins essential for terminal biosynthetic reactions of coenzyme Q10 has been an obstacle in this area. COQ5 protein has been found to be essential as one of the nine COQ proteins essential for coenzyme Q6 biosynthesis in yeast, but its role in humans for biosynthesis of coenzyme Q10 has not been studied. Yeast COQ5 protein is a methyltransferase and a mitochondrial matrix protein associated with inner membrane that can form a multi-subunit complex with other COQ proteins in yeast (Baba et al., J Biol Chem 279: 10052, 2004). However, there has been no study on human COQ5 protein.
For making polyclonal antibody against nuclear DNA-encoded mitochondrial matrix protein by using recombinant protein as the antigen, most people just expressed full-length protein in E. coli. However, that should result in misfolded protein and formation of inclusion body because removal of mitochondrial targeting signal (MTS) by peptidase in mitochondria after import of mitochondrial proteins into matrix is necessary for correct folding of the protein into mature structure and bacteria do not have such machinery (Chacinska et al., Cell 138: 628, 2009). We have previously tried to purify His-tagged full-length human COQ5 protein expressed in E. coli, but it formed inclusion under all conditions commonly employed. We could only purify that recombinant protein under denaturing condition with 8 M urea, but protein precipitated severely once urea was removed by buffer exchange with phosphate-buffer saline (PBS). Nevertheless, one publication has compared and indicated that removal of N-terminal MTS of mouse endonuclease G, a mitochondrial matrix protein, could increase the solubility of the protein when its mutant was expressed in E. coli although no data were shown for the comparison (Yoon et al., Acta. Crystallogr. Sect. F. Struct Biol. Cryst Commun 65: 504, 2009).
So far, other than Baba's article on yeast COQ5 (Baba et al., J Biol Chem 279: 10052, 2004), no publication has reported the purification of COQ5 protein for generation of antibody. Although they indeed expressed mature form of recombinant yeast COQ5 protein without N-terminal NITS in E. coli, denaturing conditions with the use of urea followed by SDS-PAGE purification were still applied for purification of recombinant yeast COQ5 proteins. The antibody produced by this group against such denatured COQ5 protein was not affinity-purified either (Baba et al., J Biol Chem 279: 10052, 2004). These results indicated that additional strategy might be needed to purify yeast COQ5 protein as soluble protein. Because yeast COQ5 protein is not a soluble matrix protein but is associated with other protein on mitochondrial inner membrane as an insoluble complex (Baba et al., J Biol Chem 279: 10052, 2004), we suspected that even if MTS-truncated could be expressed as native form in the cytoplasm of E. coli, it might become associated with plasma membrane of bacteria to form insoluble complex. The rationale is that mitochondria in modern eukaryotes is from ancient bacteria during evolution and so biochemical characteristics of mitochondrial proteins in mitochondrial matrix and inner membrane are often similar to that in cytoplasm and plasma membrane of bacteria, respectively (Pallen, Trends Microbiol 19: 58, 2011).
The earliest commercial antibodies for human COQ5 available were from Abgent (Cat.#: AP9319c) and ProteinTech (Cat.#: 17453-1-AP), although more commercial antibodies came out in 2012 with similar description as that in datasheets of these two antibodies. By using information from online datasheets of these two antibodies from Abgent and ProteinTech as examples, we could find many problems for those commercial antibodies. For example, they did not prove the detection of endogenous human COQ5 protein by these antibodies, but just used mouse tissues as demonstration. No validation experiments, such as overexpression or knockdown of COQ5 gene in cells, were performed to confirm the specificity either. Moreover, the size of protein detected by Western blot by antibodies from different sources was not the same. On the other hand, the antigen of ProteinTech's antibody, as His-tagged full-length human COQ5 protein containing MTS, is also a commercial product (Cat. #: ag10203). Although no description about the purification condition could be found on the datasheet of the antigen, it appears that this purified recombinant protein is not soluble in aqueous solution without ionic detergent as the protein is resolved in PBS containing the detergent Sarcosyl (N-laurylsarcosine) at the concentration of 0.7%.